Three anaerobic protozoan parasites, Giardia Lamblia, Trichomonas vaginalis and Tritrichomonas foetus, which all grow naturally by adhering to host epithelial cells and performing phagocytosis, have been found lacking the capability of de novo synthesis of purine and pyrimidine nucleotides and deficient in dihydrofolate reductase and thymidylate synthetase activities. They depend on rather simple salvage pathways for purines, pyrimidines and nucleosides. These pathways, delineated by us during the past two years, have presented several pivotal enzymes which not only are essential for survival of these parasites but also exhibit unique substrate specificities qualifying themselves as potential targets of antiparasitic chemotherapy. These enzymes are the APRT, GPRT and UPRT in G. lamblia, HXGPRT, UPRT and IMPDH in T. foetus and the deoxyribonucleoside phosphotransferase of T. vaginalis. The GPRT, HXGPRT and IMPDH have been purified to homogeneity and shown to have many different properties from the related host enzymes. We are planning on a screening program for inhibitors of these isolate enzymes as one way of finding chemotherapeutic agents. On the other hand we will prepare pure samples of G. lamblia GPRT, T. foetus HXGPRT, T. foetus IMPDH and mouse sarcoma 180 IMPDH in microgram quantities by the established procedures. The N-terminal sequences of the four purified enzymes will be determined, the corresponding oligonucleotides will be synthesized and used as probes to begin identifications, clonings and sequencings of the genes encoding these enzymes for sequence comparisons. It is anticipated that sequence differences associated with the enzyme active sites may explain the different substrate specificities and help new drug designs. Meanwhile, further purifications of the UPRT's and the T. vaginalis deoxyribonucleoside phosphotransferase will be pursued with affinity column chromatographies. An exciting observation of the presence of a double-stranded RNA in T. vaginalis, the discovery of the RNA gene in T. vaginalis DNA and the possible relationship between the RNA and the metronidazole-sensitivity of T. vaginalis will lead us into further pursue of these new findings. Genomic libraries of T. vaginalis will be established and the double-stranded RNA will be used as a probe to initiate the identification, cloning and sequence determination of its encoding genome for further understanding of the biological function of the double-stranded RNA.